A number of both small and large molecular products, such as antibiotics, monomers for polymeric products, amino acids, organic acids, vitamins, proteins, nucleic acids and the like, can be produced in both cell free and cell culture systems employing biochemical metabolism or reactions. These products include what is also often referred to as biologics, in contrast to chemically manufactured products, though biologics (which include also biosimilars) is often only referred to in order to describe proteinaceous pharmaceuticals.
However, usually, especially in cell culture with living cells, the presence of oxygen is necessary, at least where aerobic organisms are used.
Cellular respiration constantly generates small but significant amounts of reactive oxygen species (superoxide, hydrogenperoxide, hydroxylradicals) that are toxic and can damage cellular constituents (DNA, proteins, lipids). Negative effects of stress in fermentation processes can lead to                Decreased viability of the cells and thus shortening of the process duration,        Decreased productivity of the cells,        Damage to the products.        
Cells have developed oxidative stress protection and repair systems during evolution. However, these defense mechanisms may not be sufficient under conditions of High Cell Density Cultivation (HCDC) and in addition require cellular energy that is no more available for product formation. Especially oxidative stress is pronounced in HCDC as high oxygen input into the cultures is needed in order to sustain growth and productivity of cells at high densities. Also in HCDC cellular metabolites and by-products are formed at elevated rates which can impair growth, productivity and product stability.
Production systems for recombinant therapeutic proteins or other products are essentially classified into mammalian cell cultures and microbial systems (bacterial, yeast, fungi):
Mammalian cellcultureBacterial systemsProductivity10 g/L10-50 g/LCostHighLowGeneration of production cell6 months1-2 monthslinesProcess cycle timeWeeksDaysComplex modifications (e.g.YesLimitedglycosylation, disulfide bond(no glycosylation)formation, acylation)
A large difference between bacterial and mammalian culture systems is that bacterial growth in a nutrient solution closely resembles their natural life style while growth of suspended mammalian cells has to be considered artificial compared to the natural situation viz. growth in tissues as part of a multi-cellular organism. For mammalian cell culture systems the current trend is to replace complex animal derived media components such as calf serum with chemically defined ones. As complex media contain a large variety of components which among others have anti-oxidative activities, their omission in defined media creates a need for other stress relieving additives.
Oxygen as such, for example, can cause oxidative stress via a number of mechanisms and oxygen species. For example, dimolecular, ground state oxygen, is a free radical, can react readily with carbonyl radicals to form the organic peroxyl radical, thus leading to undesired reactions.
Oxygen can be present as singlet oxygen which exists in two states. The more reactive singlet oxygen state, O2 1σg , is a radical that contains two unpaired electrons of opposite spin in separate π anti-bonding orbitals. It can be created by reactions with porphyrin or flavins in the presence of light and can react with organic conjugated double bonds to form endoperoxides, dioxetanes and hydroperoxides and peroxides and with organic sulfides to produce sulfoxides. Superoxide contains an additional unpaired electron. It is a radical and can also be formed by several mechanisms in vivo. Transition metals such as iron and copper in the reduced form catalyze a set of reactions that result in the formation of hydroxyl free radicals, another reactive species including oxygen. Hydroxyl free radicals are formed in vitro and in vivo. A well described source for hydroxyl radicals in cells is the Fenton reaction in which hydrogen peroxide reacts with Fe(II) ions to form hydroxyl radicals and OH−The hydroxyl free radical is an extremely reactive oxidizing species. It causes damage to all classes of bio-molecules, but one of its most damaging immediate effects is the initiation of lipid peroxidation. Lipid peroxidation is a self-propagating event that is mediated by the organic peroxyl radical. Organic peroxyl radicals are formed when allylic carbonyl radicals bind ground-state oxygen. This is an extremely important reaction in vitro and in vivo. The primary molecules that undergo this chemistry are the polyunsaturated fatty acids (PUFAs). Allylic carbonyl radicals are generated when hydroxyl free radicals abstract a hydrogen atom from the allylic carbon. This produces an organic peroxyl radical that participates in a chain reaction of lipid oxidations that lead to cell membrane damage and cell death. Peroxynitrites can be produced by certain cells which produce extracellular nitric oxide as a cell-signaling molecule. Superoxides can react with nitric oxide to from peroxynitrites. Peroxynitrites react very rapidly with carbon dioxide to form carbon monoxide and nitric dioxide radicals.
In general, oxidative stress and other radical reactions can lead to damage at molecules such as proteins, nucleic acids or other small or large molecular weight molecules such as lipids, amino acids, sugars or the like, e.g. at double bonds, sulphur etc. (see e.g. Dean et al., Biochem. J. 324, 1-18 (1997) or Konz et al., Biotechnol. Prog. 14(3), 292-409 (1998)).
Measures taken in the art to prevent and to cope with oxidative and other stresses in protein production processes include    1. Natural Response to Oxidative Stress
Although not the matter of human intervention, the natural protection of cells to oxidative stress could be considered as a benchmark. Reactive oxygen species (ROS) are constantly present in cells in low concentration and in elevated concentrations during various stress conditions. Cells protect themselves from damage through ROS by enzymes that detoxify these (superoxide dismutase for superoxide, catalase and several peroxidases for hydrogen peroxide and other hydroperoxides) and by producing cellular antioxidants such as vitamin C and E as well as thiol-containing peptides and proteins (glutathione, thioredoxin) that react with ROS. However conditions applied in the production of proteins by fermentation (high cell densities, high oxygen input, major stress through massive overexpression of one specific protein) may lead to a saturation of these pathways. In addition the cellular repair of oxidative damage draws away metabolic energy (in the form of low potential reducing equivalents) which otherwise is available for product formation.    2. Use of Natural and Synthetic Antioxidants:
Natural antioxidants (Vitamins C & E, polyphenols, amino acids such as cysteine and methionine, peptides such as gluthatione and carnosine) scavenge reactive oxygen species (ROS) and are used in medicine, personal care and nutrition as well as in fermentation processes. There are also examples of synthetic antioxidants and radical scavengers being used in fermentation, however to our knowledge the use of nitroxide, HALS type molecules or phenolic antioxidants for increasing the productivity of cells in fermentation has never been described. There are also references (mostly from journals, see following page) describing that nitroxides and related substances can diminish the level of oxidative stress in mammalian cell culture and thus increase cellular lifetime by inhibiting programmed cell death (=apoptosis). However this effect seems to be cell line specific and dependent on the conditions as there are also reports that nitroxides can also induce apoptosis. Common media supplements that delay apoptosis are growth factors (insulin-like growth factor, transferrin), amino acids and peptides that specifically inhibit enzymes involved.
Antioxidants have been added to cells in culture to mitigate the deleterious results of radical formation, such as by oxidative stress, for the cells. Among these antioxidants, compounds such as ascorbic acid (see e.g. U.S. Pat. No. 3,703,439, use in L-DOPA production, not mentioning antioxidative effect) or other antioxidants (see, e.g., U.S. Pat. No. 3,704,205, use of amines or phenols (including BTH or aminophenols) in the manufacture of L-Glutamic acid in aerobic bacteria cultures; or U.S. Pat. No. 6,521,443, use of phenols or SH-group comprising compounds as antioxidants to protect lactobacilli in culture) have been described.
Also, for example, EP 1 764 415 shows the manufacture of p-hydroxybenzyl alcohol (p-HBA) by host cells in a medium to which, inter alia, antioxidants are listed as possible ingredients to be added, without, however, naming or exemplifying specific antioxidants.
U.S. Pat. No. 3,235,467 mentions certain amides, imides or lactames as activators for the biochemical manufacture of β-carotene, to mention an example where other additives are used to increase production in cell culture systems.
US 2003/0096414 mentions cell culture media allowing to transform cells by introducing nucleic acids. Among possible additives, 2-hydroxypyridine-N-oxide is mentioned as a transferring replacement compound, that is, as iron complex former.
US 2003/0114358 mentions trimethylamine-N-oxide as inhibitor of proteolytic protein degradation by hydrolyses, such as proteases, for isolated products.
US 2005/0100994 mentions the cell free enzymatic production of dietary sterol fatty acid esters in the presence of vitamin E or tea polyphenol as antioxidants.
US 2007/0053871 mentions pharmaceutical formulations of proteins, e.g. antibodies, also allowing the addition If antioxidants as preservatives, naming inter alia trimethylamine-N-oxide.
WO2008/100782 describes a fermentation process for the production of coenzyme Q10L in the presence of cysteine, ascorbic acid, dithiothreitol, glutathione and thyoglycolic acid.
WO2008/047113 discloses ethanol production with addition of radical scavengers to cell culture.
US2008/200548 makes use of N-acetylcysteine amide (Nac Amide) for Treatment of Oxidative Stress associated with infertility.
US 2007/110743 mentions the production of proteins in cell culture in the presence of anti-senescence compounds, such as carnosine or analogues thereof.
US 2007/034198 mentions the sterilisation of proteins in the presence of various antioxidants.
Other references mentions e.g. glutathione, cystein or N-acetylcystein as antioxidants.
Hirudin yield and quality in P. pastoris is increased by including 4-10 mM Ascorbic acid into the medium (see Xiao, Appl. Microbiol. Biotechnol. (2006) 72: 837-844).
Sterically Hindered and therefore metastable Nitroxide radicals, such as the 2,2,6,6-Tetramethyl-1-piperidinyloxy radical (TEMPO) and 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy have already been examined in few single cases as additives to cell culture. US 2004/024025, for example, describes that TEMPO causes increased apoptosis in human cancer cells due to SAPK and p38 MAPK signal pathway modulation, leading to protein phosphorylation. Thus results available are rather disencouraging from adding sterically hindered nitroxyls to cell cultures.
U.S. Pat. No. 5,462,946 mentions the use of various nitroxide compounds as antioxidants capable of protecting cells, tissues, organs and whole organisms against the deleterious effects of harmful oxygen-derived species generated during oxidative stress in pharmaceutical formulations. It does not specifically refer to the use in the manufacture of products in cell culture or by biosynthesis in cell free systems. Among many other possible uses, it is mentioned only that they can be used in stabilizing labile chemical compounds which undergo spontaneous degradation by generating free radicals in media which are already present in the media as such.
US 2008/305055 mentions the use of sterically hindered nitroxyl and certain specific phenol compounds as drugs in the treatment of inflammatory or related conditions in humans.
Bednarska, S., et al. described the use of various antioxidants (e.g. ascrorbic acid, cysteine, glutathione, N-acetylcysteine, dithiothreitol, trolox, quercetin, melatonin, TEMPO, phenyl butylnitrone) in the protection of protein thiols of proteins within Saccharomyces Cerevisiae. 
Other approaches to optimize yield in biosynthetic production processes include:    3. Use of Growth Factors, Cytokines and Other Growth Stimulating Molecules as Additives
The use of hormones, other signalling molecules and small molecule growth stimulating compounds is most common in mammalian cell culture as these cells grow in vivo within tissues and communicate with surrounding cells through chemical signals. As the use of calf-serum as a source of growth factors in mammalian culture media is less and less accepted for production purposes there is a tendency to include specific growth factors which are mostly proteins from defined non-animal sources. Examples for growth factors are insulin, insulin-like growth factor or epidermal growth factor. Also used are interleukins, cytokines such as interferon, and transport proteins (e.g. albumin, transferrin). All of the aforementioned substances are proteins. In addition are vitamins, amino acids and lipids are also used. (For a general reference see Wove R A 1993 Media for Cell Culture, Biotechnology, Wiley-VCH, 2nd Ed., Vol 3, p. 141-156)    4. Engineering of Cell Lines that Show Greater Stress Resistance and Longer Lifetime
This can be achieved either through random mutagenesis or rational introduction of benefical genes. Examples of genes that add to cellular fitness are those that have antioxidative activity such as superoxide dismutase and catalase and genes that contribute to the synthesis of cellular antioxidants such as glutathione biosynthesis. Cellular lifetime is increased by preventing the cells from entering into programmed cell death, also termed apoptosis. Apoptosis, is a regulated physiological response resulting from a non-lethal stimulus that activates a cellular cascade of events culminating in cell death (Arden 2004 Trends Biotechnol 22: 174-180). Increases in cellular fitness and lifetime has been obtained through either both random mutagenesis and subsequent selection of improved cells or through specific over-expression of genes that confer higher viability and lifetime.    5. Process Optimization to Minimize Detrimental Conditions
Major factors considered in process optimization are (1) to achieve high cell densities, (2) longer lifetime of the producer cells, (3) optimal feeding strategies, (4) optimal supply of gaseous substrates, especially oxygen, and (5) media composition.
None of the prior art disclosures ever suggested the use of nitroxiles and related compounds alone or in combination with other antioxidants such as specific phenols in biosynthetic methods using cell culture or cell free biosynthesis systems to obtain small and/or large molecular biosynthetic products, that is, in the manufacture of such products.
There is an increasing lack of capacity for sufficient production of biotechnologically manufactured products which in some cases already leads to anticipated bottlenecks, e.g. where eukaryotic cell culture systems are used.
It is thus important to find ways to increase the quality and the quantity or both of products obtained in biosynthetic processes.